Development of a versatile high-throughput mutagenesis assay with multiplexed short-read NGS using DNA-barcoded supF shuttle vector library amplified in E. coli.

A forward mutagenesis assay using the supF gene has been widely employed for the last several decades in studies addressing mutation frequencies and mutation spectra associated with various intrinsic and environmental mutagens. In this study, by using a supF shuttle vector and non-SOS-induced Escherichia coli with short-read next-generation sequencing (NGS) technology, we present an advanced method for the study of mutations, which is simple, versatile, and cost-effective. We demonstrate the performance of our newly developed assay via pilot experiments with ultraviolet (UV) irradiation, the results from which emerge more relevant than expected. The NGS data obtained from samples of the indicator E. coli grown on titer plates provides mutation frequency and spectrum data, and uncovers obscure mutations that cannot be detected by a conventional supF assay. Furthermore, a very small amount of NGS data from selection plates reveals the almost full spectrum of mutations in each specimen and offers us a novel insight into the mechanisms of mutagenesis, despite them being considered already well known. We believe that the method presented here will contribute to future opportunities for research on mutagenesis, DNA repair, and cancer.

Burrow Morphology of Alpheid Shrimps: Case Study of Alpheus brevicristatus and a Review of the Genus.

The Alpheus brevicristatus De Haan, 1844 is one of the commonest shrimp species inhabiting the tidal flats in Japan. This species is sometimes accompanied by the facultative symbiotic goby, Acentrogobius spp. Here, we investigated the burrow morphology of A. brevicristatus in a tidal flat of Uranouchi Inlet, Kochi Prefecture, Japan. We also reviewed existing literature on alpheid burrow morphology using the resin casting technique, to determine how burrows vary in the presence and absence of gobies. Nine burrows were casted in situ using polyester resin. All burrows were long, but shallow in structure, with several funnel-shaped openings and short cul-de-sac branches. This species appears to use several burrow openings to access the sediment surface for feeding with high efficiency. Gobies were not associated with all burrows cast; however, 1-3 individuals of the small alpheid shrimp Athanas japonicus Kubo, 1936 were entombed in seven of the casts. A review of 12 studies on the burrow morphology of 16 Alpheus species based on resin casting techniques showed wide variation in burrow characteristics, such as burrow depth, length, and number of openings. Our findings suggest that burrow structure is influenced by species-specific characteristics and sediment type. The possibility that the presence of the symbiotic goby affects the burrow morphology of Alpheus shrimp is discussed.